PROJECT SUMMARY/ABSTRACT Hydrogenases are a class of complex metal containing enzymes that generate energy for certain organisms by catalyzing reversible interconversion between protons and hydrogen gas. Unraveling the intricate mechanistic details about the mode of action of this enzyme could lead to significant advances in the alternate energy research. However, the innate complexity of these enzymes due to the presence of many metallic cofactors, low yield of purification and oxygen intolerance makes studying these enzymes challenging. Our long term goals are to design artificial hydrogenases that serve as simpler platforms mimicking these metalloenzymes. Metalloprotein design is an appealing and well-established approach to model complex metalloproteins in nature using minimal protein scaffolds. Although these designed systems are less complex than native enzymes, they can serve as structural and functional analogues of the native metalloenzymes allowing rational engineering of both the primary and secondary shell interactions within the protein scaffold. Employing this approach, and guided by strong preliminary data, we propose to pursue two Specific Aims describing the overall design principles of artificial hydrogenases. Furthermore, we will investigate how the metal site and surrounding protein scaffold work synergistically to influence the physical and catalytic properties of the designed metalloenzymes. The Specific Aims are: 1) Reengineering a thiolate-rich copper storage protein (Csp1) into a Ni binding protein (NBP); and 2) Elucidate the role of remote interactions beyond primary coordination in fine-tuning the thermodynamic, redox, and catalytic properties of NBP. Our data indicate that the designed NBP is folded and stable in solution, and demonstrates highly cooperative unfolding behavior. NBP binds 1 equivalent of Ni(II) with micromolar affinity. In addition, Ni(II)-NBP is also active for electrocatalytic and photocatalytic H+ reduction to H2 gas. Collectively, results obtained from the proposed research will broadly impact the fields of metalloprotein design, bioinorganic chemistry, and alternate energy research. Our studies will establish new principles in rational design of artificial hydrogenases, and provide insight into the likely functional mechanism of these metalloenzymes. Using this knowledge, it would be possible to prepare new biocatalysts with improved stability and novel functions.